• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.52-52
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• 2002

The objective of this study was to determine the developmental competence of in vitro matured bovine oocytes after intracytoplasmic sperm injection(ICSI) with frozen-thawed epididymal spermatozoa. The ovaries were obtained from slaughtered Korean native cows. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with frozen-thawed epididymal spermatozoa. After ICSI, one group of oocytes was activated with 7% ethanol for 5 min, and second group was not activated. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24-30 hrs in a incubator with 5% CO₂ in air at 38.5℃. (omitted)

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.55-59
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• 2002

The objective of this study is to determine the developmental competence of in vitro matured bovine oocytes after intracytoplasmic sperm injection(ICSI) with frozen-thawed epididymal spermatozoa. The ovaries were obtained from slaughtered Korean native cows. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with frozen-thawed epididymal spermatozoa. After ICSI, a group of oocytes was activated with 7% ethanol fur 5 min, and the other group was not activated. The oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24~30 hrs in a incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. The percentage of oocytes reaching M II after 24 hrs and 30 hrs of incubation were significantly higher(p<0.05) after culture with TCM-199 media(80.0% and 88.3%) than M I(8.3% and 6.7%). The rate of cleavaged embryos to blastocyst obtained by ICSI treated activation oocytes was significantly higher(p<0.05) than that of nonactivation oocytes(22/46, 47.8% vs 10/39, 25.6%). The rates of embryos development to blastocyst obtained by ICSI treated sperm of flesh, epididymal and frozen-thawed epididymal were 24/45(53.3%), 15/40(37.5%), 11/43(25.6%), respectively and these values of fresh sperm injection were higher than frozen-thawed epididymal sperm. We also concluded that embryos can be produced with ICSI of in vitro matured oocytes by ICSI using frozen-thawed epididymal semen.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.155-159
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• 2004

This study was carried out to investigate the general characteristics and viability of sperm after freezing and thawing and the pregnancy rates after artificial insemination with thawed semen. The rates of viable sperm after slow and rapid freezing were 87.4±3.85% and 70.8±4.45%, respectively which were significantly lower than that of fresh semen control (91.7±3.45%). The mean concentration of epididymal sperm after dilution in 1.0 ml saline and. 3.0 ml extender in a various concentrations of cryoprotectants was 124.5±48.3 x 10/sup 6/ (range of 45 x 10/sup 6/ to 280 X 10/sup 6/ /ml). There was a significant difference not in the percentage of acrosome-reacted sperm, but in the percentage of capacitated sperm, between fresh and frozen-thawed epididymal semen. When frozen-thawed after diluting with tris-buffer extender containing glycerol, DMSO and ethylene glycol with concentration of 2 to 6%, the rates of epididymal sperm exposed to different cryoprotectants ranged from 14.4±4.7% to 20.7±5.8%, 17.8±5.2% to 36.5±4.9%, and 14.4±4.6% to 18.5±5.3%, respectively which were lower compare to fresh semen control. The pregnancy rate after artificial insemination with frozen semen was 70.6%, whereas that with fresh semen was 90.0% in dogs with naturally induced estrus.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.271-276
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• 2015

The aim of the study was to investigate the ability of sperm derived from the epididymis in regard to sperm motility, sperm penetration to oocyte and subsequent development of the embryo. Frozen-thawed sperm from epididymis showed similar percentage of motile sperm (VSL ${\geq}25{\mu}m/sec$) as compared to that of commercial sperm (control). Sperm penetration of frozen-thawed epididymal and commercial sperm was not significantly different. Moreover, cleavage and blastocyst rates were similar in both epididymal and control. Sperm derived from the epididymis also showed fertilizability and subsequent embryonic development.

• Korean Journal of Animal Reproduction
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• v.21 no.3
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• pp.247-253
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• 1997

The objective of this study was to examine the fertilization and embryonic development rates of porcine oocytes matured in vitro according to the morphological normality of epididymal sperm in porcine IVF. The results obtained in this experiment were summarized as follows : 1. When the ratio of morphological normality of epididymal sperm was divided into the three groups with $\leq$ 10%, 10-30% and $\geq$ 50%, the fertilization and embryonic devel-opment rates of 능50% group (64%. 26%) w were significantly higher than those of other two groups ($\leq$10%: 27%, 6% and 10-30%: 36%. 5%) (p$\leq$50% group of morphologically normal epididymal sperm was adjusted to 100% (5 x 10$^5$ cells/ml), the fertilization a and development rates (63%, 27%) of epidymal sperm were similar to those of frozen-thawed ejaculated sperm (56%, 35%). 3. Also, when the fertilization and development rates of epididymal sperm were analyzed according to the oocyte : sperm ratio (1:6000, 1: 6650, 1: 7700, 1: 10000), the fertilization and development rates indicated high, in 1:6000 (68%, 32%) and 1:6650 (89%, 31%), the ratio of oocyte: sperm. Therefore, when the percentage of morphological normality of epididymal sperm is more than 50, the embryonic development a can be obtained similar to that of frozen thawed ejaculated sperm. Also, these result suggested that the morphological evaluation of normal sperm in porcine IVF using epididymal sperm sho비d be prerequisite for the more effective embryonic developm ment.

• Korean Journal of Animal Reproduction
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• v.21 no.3
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• pp.239-246
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• 1997

The objective of this study was to evaluate the variation of fertilizing ability following the morphologically normal sperm ratio in porcine IVF using epididymal sperm The results obtained in this experiment were summarized as follows: 1. When the penetration rate (PR), polysper my rate (PSR), pronuclei formation (2PNF) and mean number of sperm (MNS) per oocyte were evaluated according to the percentage of morphologically normal epididyrnal sperm at insemination($\leq$lO%, 10~30% and $\geq$50%). the PR and PSR of $\leq$50% group (82.4, 87.4%) were significantly higher than those of other two groups ($\leq$lO%; 29.7%, 22.6% and 10~30%; 20.3, 37.0%) (p<0.01). Also, the 2PNF per examined oocytes was significantly high in $\geq$ 50% group (p<0.01). 2. When the $\geq$50% group in epididymal sperm was adjusted to 100% (5x1$^5$ cells/ml) , the PSR and 2PNF were not different between epididymal sperm (86.7, 35.1%) and frozen-thawed ejaculated sperm (86.0. 39.4%) although the PR in epididymal sperm (79.7%) was significantly lower than that in frozen-thawed ejaculated sperm (95.5%)(p<0.01). 3. Also. when the PR, PSR, 2PNF and MNS of epididymal sperm were evaluated according to the oocyte: sperm ratio (1:6000, 1:6650. 1:7700 and 1: 10000) at insemination. the PR, PSR and MNS were increased as the oocyte:sperm ratio increases. However, this result indicated that the 2PNF was high in the oocyte:sperm ratio (1:6000 and 1:6650). Therefore. these results suggested that when the percentage of morphologically normal epididymal sperm was more than 50. the fertilizing a ability was very similar to that of frozen-thawed ejaculated sperm and that the detailed evalu¬a ation of morphological normality in porcine IVF using epididymal sperm should be prerequisite to obtain the more effective fertilizing ability.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.297-302
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• 2013

Cryopreservation of epididymal spermatozoa offers a potential tool for rescuing genetic material from males of genetically elite populations. Castration, catastrophic injury, sudden death or any other event that makes semen collection or mating impossible may prematurely terminate a stallion reproduction. Stallion epididymal spermatozoa vary widely in the loss of progressive motility, acrosomal integrity, and viability during freezing and thawing. The objective of this work was to investigate the effect of (1) freezing package types on cryopreservation efficiency, (2) thawing temperatures (37, 56 or $70^{\circ}C$) on Computer Assisted Sperm Analysis (CASA) parameters and (3) post-thawing incubation time (0, 1, 2 or 4h) on castrated stallion epididymis. Post-thawed sperm motility ranged between 59.69% and 64.28% ($56^{\circ}C$ and $37^{\circ}C$) in various thawing temperatures. When stallion epididymis sperm was frozen, straw was better than freezing tube on VCL (Velocity of Curvilinear Line) and VAP (Velocity of Average Path) parameter. Higher percentage of motility was observed at $37^{\circ}C$ thawing temperature even though no significant difference was observed among various temperatures. The motility, VCL, ALH (Amplitude of Lateral Head displacement), VAP, BCF (Beat-Cross Frequency) and STR (Straightness index) parameter of post-thawed sperm were significantly decreased by increasing the incubation time for all thawing temperatures. The present study showed that type of freezing package (Straw vs. Freezing tube) was not significantly different on cryopreservation efficiency. Furthermore, stallion epididymal spermatozoa frozen-thawed at $37^{\circ}C$ for 1 min resulted the highest proportion of motility and velocity movement. In addition, motility and viability of frozen-thawed stallion epididymal spermatozoa were also decreased by incubation.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.105-110
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• 2002

The objective of this study was to determine the developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection(ICSI) with epididymal spermatozoa. The ovaries were obtained from slaughtered small species dogs. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with epididymal spermatozoa. After ICSI, one group of oocytes was activated with 2.0 mM dimethylaminopurine or 7% ethanol for 5 min. and second group was not activated. The follicular oocytes were cultured in synthetic oviductal fluid(SOF) and TCM-199 medium containing hormones and 10% FCS for 24~48 hrs in a incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. 1. Results of IVM showed that the percentage of oocytes reaching MII after 24 h and 48 hrs of incubation were significantly higher(p<0.05) after culture with 48 hrs(9/30, 30.0%) than that after culture with 24hrs(a/30, 26.7%). 2. Results of IVM showed that the percentage of oocytes reaching MII after 48 hrs of incubation were significantly higher(p<0.05) after culture with SOF media(10/30, 30.3%) than TCM-199 media (7/30, 23.3%). 3. The rate of cleavaged embryos to blastocyst obtained by ICSI treated activation oocytes was significantly higher(p<0.05) than that of nonactivation oocytes(5/16, 25.0% vs 1/13, 5.0%). 4. The rates of development of cleavaged embryos to blastocyst obtained by ICSI treated sperm of fresh, epididymal and frozen-thawed epididymal were 8/18(44.43%), 5/16(31.3%), 2/14(14.3%), respectively. and these values of frozen-thawed epididymal sperm injection were lower than fresh sperm injection.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.321-326
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• 2018

In this study, two epididymal spermatozoa recovery methods in relation to sperm number, motility, viability and acrosome reaction were examined. Seven bulls were castrated and 7 testicles with epididymides were transferred to the laboratoy. Epididymis in each bull was randomly used for flushing and mincing methods with semen extender (Optixcell, IMV, France). The recovered spermatozoa with adjusted sperm concentration to $40{\times}10^6cells/mL$ was diluted with optixcell and cryopreserved. In experiment 1, the difference in the total number of spermatozoa using flushing and mincing methods was insignificant (2570.0 and $2505.2{\times}10^6cells/mL$, respectively). For experiment 2, the percentage of motile spermatozoa and motility parameters between flushing and mincing methods were studied through the use of sperm class analyzer after frozen-thawing. The percentage of total motile sperm between flushing and mincing methods was almost the same with $89.5{\pm}12.8$ and $91.4{\pm}7.9%$, respectively. The same is the case with experiment 3 wherein the viability and acrosomal integrity of frozen-thawed epididymal spermatozoa by flushing and mincing was insignificantly different. The results from the study showed that both flushing and mincing methods can be used for epididymal spermatozoa recovery in bull.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.229-234
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• 2002

This study was carried out to investigate the general characteristics such as concentration sperm motility and abnormality of sperm on the whole epididymal semen(EWS), RSP-S(removed seminal plasma by saline) and RSP-T(removed seminal plasma by tris-buffer) semen and survival rates after freezing on motility of whole and RSP-S and RSP-T semen and extender containing 2~8% glycerol, and ability of frozen-thawed sperm to penetrate homologous oocytes. 1. The concentration, motility and abnormality of epididymal WES, RSP-S and RSP-T sperm were 4.25 $\pm$ 0.25($\times$10$^{6}$ Cells/$m\ell$), 3.85$\pm$0.20($\times$10$^{6}$ Cells/$m\ell$), 4.05 $\pm$ 0.28($\times$10$^{6}$ Cells/$m\ell$), 50.55 $\pm$ 2.75%, 67.25 $\pm$ 2.55%, 78.75 $\pm$ 3.55 and 9.45 $\pm$ 2.25%, 37.75 $\pm$ 2.10%, 24.25 $\pm$ 1.55%, respectively. 2. The survival rates of slow and rapid frozen epididymal RSP-S and RSP-T sperm were 35.00 $\pm$ 2.35%, 45.50 $\pm$ 2.15％ and 16.50 $\pm$ 3.55%, 22.55 $\pm$ 3.95%, respectively. The survival rate of epididymal WES and RSP-T sperm after freezing following dilution with tris-buffer containing 2~8% glycerol were 9.25 $\pm$ 1.55%~17.50 $\pm$ 2.50%. 3. The percentage of capacitated and acrosome-reacted sperm prier to culture for fresh and frozen -thawed epididymal RSP-T semen were 45.25 $\pm$ 5.75%, 7.06 $\pm$ 0.25%, 48.20 $\pm$ 6.80% and 13.00 $\pm$ 2.35%, 3.55 $\pm$ 0.85%, 15.50 $\pm$ 1.90%, respectively. The penetration rate the number of sperm per penetrated for fresh and frozen-thawed epididymal RSP-T sperm were 39.25 $\pm$ 4.72%, 34.24 $\pm$ 3.93% and 1.30 $\pm$ 0.33, 1.10 $\pm$ 0.50., respectively.